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Friday, March 28, 2014

Time and again, in this blog, I have written about the effects of microbiome. One of the earliest concepts in field of microbiome, by competing with other pathogenic organisms they provide the earliest level of defence. That is one of the reason why our immune system is tolerant to them. When I say "Microbiome", most of the papers to date published, reflects the bacterial population. There are relatively literature on fungal microbiome.

HIV doesn't need any introduction in this webpage. In clinical context, HIV is usually suspected when they make an appearance with infections of low pathogenic organism. In other words, features suggestive of immuno-compromised state. There is a whole range of list, of what the organism can be. But, there are a couple of signature organisms- Oral thrush by Candida species, Cryptococcal meningitis, Respiratory infection by pneumocystis jiroveci. In this blogpost, we will consider Candida infection.

Just have a look at Photo 1. The photo depicts how bad a oral candidiasis can be. There is very sparse literature, on distribution of Candida species in healthy population. There is probably a huge variation. C albicans, is seen in a good number of healthy individuals. So why does candida become a problem in HIV infected patients. The most obvious explanation is that the HIV reduces immune barrier and so candida doesn't have to fight it out. Exactly how does a candida decide that its time to attack, is not known. There has been a lot of speculation in this field. Eh, possibly. But there seems to be a better explanation.

There has been some earlier hints that perhaps there is a role of microbiome. A new study published in PLOS, looked at the changes in fungal and bacterial microbiome, using the metagenomic approach. By studying salivary samples from 12 HIV infected patients and 12 controls, they characterised the most common population and labelled them as COM (Core oral Mycobiome) and COB (Core oral bacteriome). The COB and COM, defines what is seen more commonly, thus excluding the variation. In the COB group 14 species where found, 13 was found common between HIV and control group. In COM, C albicans was seen in both groups. What was striking is that another member of flora, pichia was seen to negatively correlate in numbers.

On further investigation, the data unfolds that Pichia sp actively inhibits Candida, Aspergillus and Fusarium. Pichia was able to compete with candida for nutrition, inhibited virulence factors and biofilm formation. The effects was seen due to an uncharacterised molecule present in the culture supernatant medium, tentatively named as PSM (Pichia spent medium)

This is a first study of its kind. As the author suggests, there is a lot of potential in identifying the molecule so as to treat patients suffering from oral candidiasis. But, I speculate that Pichia can be a good candidate in investigating its use a probiotic. Or maybe saliva transplant is the future (Just as stool transplant is now making headlines).

Saturday, March 22, 2014

Here's a thing that you may want to try if you are in a health care center. Find out what is the most bothersome bug (I'm referring to microbes), in the hospital. Chances are you will be told MRSA (Methicillin resistant Staphylococcus aures). Notably, people have tried to come up with new drugs, vaccines or other strategies, and desperation is on the scene.

MRSA was first reported in 1961, soon after the introduction of Penicillin. But the first clinical cases were identified in USA, 1968. As per the CDC, Methicillin-resistant Staphylococcus Aureus (MRSA) is a type of Staphylococcus aures, which is resistant to certain antibiotics called beta-lactams. These antibiotics include penicillin, methicillin, oxacillin and amoxicillin. The very importance of detecting MRSA has led to development of a variety of tests. I have detailed about them in brief here. There are 2 types of MRSA depending on its primary source- HA MRSA (Hospital acquired) and CA MRSA (Community acquired).

Table 1: Contrasts between CA and HA-MRSA.

The most important question that I have been often asked about is, when MRSA has been detected, what does that mean in terms of patient care? Consider the following argument. Staphylococcus species represents an important normal flora especially in the skin of humans. S aureus is seen in a large variety of healthy people. As many as 25- 40% of people are know to carry MRSA in their body without any consequence. One of the argument is that if the person is in hospital for longtime, or is a health care worker treatment is justified. This is to avoid dissemination. I have often questioned this for the simple reason that there is enough MRSA already in the hospital and this doesn't account for extra burden. Do we need to try and treat such people thereby introducing a component of antibiotic resistance selection pressure?

Table 2: Immune based approach against Staphylococcus

Use of common procedures such as good cleanliness can get the job done in some people. Many require treatment with 2% mupirocin (for nasal carriers) or 4 % Chlorhexidine hydrochloride soap (for skin carriers). In a good number of cases this mayn't provide results and hence there is interest in developing newer strategies like use of epidermidis serine protease. Link

The problem doesn't end there. As I have said always, vaccine and immune based approaches are the best methods to combat microbes. Several companies have developed these approaches (well known products are shown in Table 2). However, most of them have failed to deliver the expected results and have been discontinued from research.

MRSA infections can be treated with drugs such as Vancomycin. Since vancomycin resistance is on slow rise several other molecules such as daptomycin, streptogramins etc have been proposed as next line of defense. However, we clearly need new antibiotics. The latest research from a group led by Mayland Chang and Shahriar Mobashery have discovered a new class of antibiotics which are potentially active against MRSA. The chemical is oxadiazole, which are Non-β-lactam Inhibitors of Penicillin-Binding Proteins. The chemical showed up in a screen of 1.2 million chemical in-silico search conducted by the researchers which on further tests showed a good activity.

At the end I want to make a digression, cause I thought this data is important in this page. Just as much as the treatment and identification of MRSA is important so is the deep seated Staphylococcus infection. Even the best of currently available methods, takes about a day for the diagnosis to be made. So if the diagnosis can be pointed right in the body in a very small turn around time that would be extremely useful. A proof of concept study was done by Frank etal. A short synthetic oligonucleotides with chemical modifications and flanked with a fluorophore and quencher was designed which could be cleaved specifically by staphylococcus aureus using micrococcal nuclease. The probe was systemically administered to a mice. The probe is activated in the areas of lesion which could be detected by imaging. Such noninvasive techniques are probably the tools of future.

To conclude, to fight MRSA we need to develop faster diagnostics, better antibiotics and maybe a vaccine that will come handy. We haven't still lost the battle

O'Daniel PI etal (2014). Discovery of a New Class of Non-β-lactam Inhibitors of Penicillin-Binding Proteins with Gram-Positive Antibacterial Activity. Journal of the American Chemical Society, 136 (9), 3664-72 PMID: 24517363

Tuesday, March 11, 2014

In the previous post, I have tried to argue over the fact as to why we wouldn't ever have a system where there is zero mutation. Ability to mutate, provides a competitive edge. I also highlighted on why mutation is favoured despite a heavy loss in terms of population number, as the case seems to be in the first place. But somebody had this question. Why didn't in that case life evolve a very high mutation rate?

Intuition says if we had a very high mutation rate, such that every new generation has a new variant wouldn't it provide a gambling advantage. The answer, though temptingly “Yes” is a “NO”. As in my previous post to argue on the reason you have to set-up another thought experiment.

Set up a culture condition, with a strain X, which has such a high mutation rate that every new progeny is a variant. Round one of replication, 2 new strains are generated with new mutations. As I said in my previous post, most of the mutations are deleterious (When I say most, I mean to say that almost 50-80% of the mutation is not compatible). Probability says that there is more chance that both the cells will not survive. And gene pool is not saved. Clearly a bad strategy. Even if the low probability event of a single strain acquiring the good mutation (or at least a life compatible mutation), the very next round of replication, it would be lost. My point is simple, there is no back up copy of the original version which can be relied upon.

This is the basis of mutation. You need to strike a balance between how much you can mutate and how much you cannot. It is important not to lose a backup copy of the original genotype. This ensures that the newly created versions (which are far less in number than the original type) need to be better and thus compete with the original type to establish itself. This ensures evolving strains are better in a given set of condition.

It is important to understand that mutation is not a universal improvement in the organism. Mutation confers the ability to survive and reproduce in a given condition only. There is a trade off (I borrowed this from R Dawkins argument; From the book titled “Greatest Show on Earth”). A bacteria that is more antibiotic resistant (by virtue of sat multiple plasmids), would have traded off some genes that are related to virulence. In other words, the strain is extremely fit to deal with strong antibiotics but less virulent. This phenomenon has been published by multiple studies. If the heavy pressure of antibiotics is lifted off, the organism may regain some virulence and lose some of the resistance plasmids. You see, it is very costly to maintain additional genes, for no reason. This is exactly the reason, why strategies such as not using certain type of antibiotics, for a sufficient period of time, gives rise to sensitive mutants or revertants.

Tuesday, March 04, 2014

Time to time, I have been receiving queries related to subject. The questions have sometimes lead to me to think deep into matters and sometimes go deep down into papers. Whatever said, it is interesting. It seems certain questions are repeated by many people. So I thought, the question needs to be addressed with my thoughts and opinion in a broader open space. That should avoid some people the trouble of framing questions for me. I have rephrased the question so as to get the readers what is being asked for

The common question is about mutation. Mutation is an important aspect of evolution. Evolution, lets 2 organism compete (for whatever survival purpose). If mutation wasn't there on first place then that should have still kept 2 different species on equilibrium. So why haven't there been an example of no mutation in the first place. In ASM blog (Link), a question was asked. Given that most mutations are deleterious, why does the mutation rate not evolve to zero?

The most common answer is "Mutation causes evolution, which sustains the need for genes to pass on, and hence mutation cannot be zero". Lets design a thought experiment. If I artificially grow a bacteria in a medium for long generations (say some 10000 generations), without any hinderance, we wil get a population that has no selective factor. So over generations, theoretically speaking mutation is leading to more wastage, thus mutation rate should decline. This is called as "General reduction principle". Such an experiment has been done (Link). The result was that mutation rate didnt decline, instead has increased by about 2 orders. The explanation is very simple. To start with, there was a mutation rate (however small that be), which created random mutants which has better properties (However slight edge that maybe) which fuelled competiton thus not allowing other members to reduce the mutation rate. This phenomenon is known as "Mutator hitchhiking hypothesis". My simple point is this, Mutation already exists at a certain level, which cannot be brought down, since there are cheaters in the field.

Fig 1: Mutation fuelling itself.

A seemingly subsequent question is how did mutation come into existence in first place. There is no clear evidence to support thge idea, but anyways idesigned one more thought experiment. Lets say I have 2 bacterial strains (X and Y) with zero mutation rate. Inoculating X and Y into a nutrient rich medium, the DNA polymerase begins to transcribe to make new copies. Polymerase of X increases the speed of incorporation of nucleotides, but Y is steady. In no time, X would be in larger quantities than Y (advantage). This selects for increasing polymerase speed till a limit is pushed where the excessive speed of incorporation leads to first mutation. The genetics can sacrifice the change in 1 base for the cost of achieving more copy numbers, and the concept of mutation is born. Once mutation has entered the scene, mutation fuels itself. Of course such an experimet can never be conducted (For many reasons, including we never have two strains of organism that has zero mutation to start with). Please note, one of the main reasons of mutation is mis-incorporation of bases due to excessive polymerase speed of work.

Now coming to a major question. Given that most mutations are deleterious, why does the mutation rate not evolve to zero? One more thought experiment (ok, this is the last). Say, I have a bacteria that produces a mutation once in every 1 generation (practically mutation rate is calculated per base incorporation. This is just for making calculations easy). That means, when the cell divides there is a mutant and a normal. And lets say the mutant is not fit for survival. The mutation is deleterious. The unmutated cell survives and replicates. Lets say this happens a million times. The cell has actually lost a million cells and by chance one mutation is good, which leads to appearance of a variation. The variation being a good one can compete with the normal type when there is a selective pressure. If there is no selective pressure, the mutation being non lethal still survives and replicates, the number being less than the normal.

Overtime (after billions of replication rounds, I have a heterogenous population. There is more than 99% of normal type, and 1% of different variants and during the process lost a billion cells in trying to be different. To this if I add an antibiotic, the mutant (which maybe less than 0.01%) survives which can now proliferate. If I had a zero mutation rate, everything would have been killed in one boom. Eventhough in the process I lost billions of cells (deleterious mutation), the remaning 0.01% of cells will carry the gene pool. Without mutation whole gene pool is lost. Given the fact that cells are exposed to all kinds of challenges, mutation provides a means to escape total anhilation even though there is significant loss (This is in contrast with total loss by trying to save more cells). This is clearly advantageous.

I hope that clarifies the question of why mutation is important and we can never have a zero mutation scenario.